差速贴壁法原代培养椎间盘髓核细胞中的应用优势  

Excellence of differential velocity adherent procedure in primary culture of nucleus pulposus cells of intervertebral disc

作　　者：胡永凯[1] 孙浩林[1] 漆龙涛 李淳德[1] 

机构地区：[1]北京大学第一医院骨科,北京市100034

出　　处：《中国组织工程研究》2016年第42期6284-6289,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81441058)~~

摘　　要：背景:髓核细胞作为椎间盘的主要功能细胞是退变机制研究的重点对象,维持体外培养的髓核细胞的生理功能及细胞表型的稳定至关重要。目的:通过对照研究差速贴壁法在犬鼠椎间盘髓核细胞原代培养中的应用优势。方法:取4周龄雄性Wistar大鼠20只,体外分离培养椎间盘髓核细胞,待原代细胞汇合近90%时进行分组传代,差速贴壁组在传代细胞贴壁30 min时,将未贴壁细胞吸出重新调整浓度后传至新的培养皿中,对照组不作处理。差速贴壁组第1、2代细胞均采用该法分离纯化。对两组第3代细胞进行细胞形态学比较及免疫组化鉴定纯度分析、CCK-8检测细胞功能活性并绘制细胞生长曲线。结果与结论:(1)倒置显微镜观察及苏木精-伊红染色,差速贴壁组细胞均一性较对照组明显增高,对照组髓核细胞中混有较多的成纤维样细胞;(2)Ⅱ型胶原免疫组化鉴定及纯度分析,2组细胞胞浆均为被染成黄褐色,证实为髓核类软骨细胞;(3)纯度分析差速贴壁组阳性率显著高于对照组(P<0.05);(4)CCK-8细胞生长曲线显示,2组细胞均经过2d的生长潜伏期,3d的指数生长期,进入生长停滞期,而对照组较差速贴壁组在潜伏期和指数增长期的早期生长更迅速。(5)结果说明,差速贴壁法是原代培养大鼠髓核细胞一种实用、有效的分离纯化方法;原代培养、经2次差速贴壁法分离纯化的第3代大鼠髓核细胞代谢旺盛、表型一致,细胞纯度更高,其对数增长期可以作为椎间盘细胞机制研究的最佳时期。BACKGROUND：As the main function cel of intervertebral disc, nucleus pulposus cel s are the focus of studying the degenerative mechanism;thereby, it is crucial to maintaining the physiological function of nucleus pulposus cel s in vitro and the stability of the cel phenotype. OBJECTIVE：To study the excel ence of differential velocity adherent procedure in primary culture of nucleus pulposus cel s of rat intervertebral disc through comparison. METHODS：Twenty male Wistar rats aged 4 weeks were enrol ed, and then nucleus pulposus cel s of intervertebral disc were isolated and cultured in vitro;cel passage culture was performed in different groups when the primary cel s were merged to 90%. Differential velocity adherent group cel s adhered for 30 minutes, and non-adherent cel s were aspirated and transferred to new culture dish after readjusting the concentration;the controls received no intervention. Passages 1 and 2 cel s in the differential velocity adherent group were isolated and purified by the same procedure. The morphology of three generations of cel s in the two groups was compared, the purity of the identification was detected by immunohistochemistry, the cel viability was detected by cel counting kit-8 and the cel growth curve was drawn. RESULTS AND CONCLUSION：Inverted phase contrast microscope and hematoxylin-eosin staining revealed that the cel homogeneity of the differential velocity adherent group was significantly higher than that of the control group, and there were more kinds of fibroblast-like cel s in nucleus pulposus cel s in the control group. Identification and purity analysis of col agen type II showed that the cytoplasm of two groups were both stained brown, indicating that they were the nucleus pulposus chrondrocytes. The positive rate of differential velocity adherent group was significantly higher than that of the control group （P〈0.05）. The cel growth curve of cel counting kit-8 showed cel s in the two groups al passed by the latency phase within 2 days, then to the logari

关 键 词：椎间盘 细胞 培养的 细胞衰老 组织工程 组织构建 软骨组织工程 差速贴壁 髓核细胞 纯化 细胞去分化 细跑老化 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]